Apparatus and method for fluorescence imaging and tomography using spatially structured illumination

ABSTRACT

An imaging system for imaging an object. More specifically, an imaging system enabling depth sectioned fluorescence imaging in a turbid medium, such as human or animal tissue, to substantially minimize the excitation radiation from reaching the detection beam path. The imaging system includes an arrangement of the excitation radiation source wherein the optical axis of the source is inclined relative to the optical axis of the camera, the optical plane of the source and the optical plane of the object are subject to a Scheimpflug condition, and the angle of inclination of the source is selected such that the excitation radiation incident upon the object is reflected to minimize excitation radiation from reaching the detection beam path.

This application is a continuation of U.S. patent application Ser. No.12/411,432 filed on Mar. 26, 2009, which is hereby incorporated byreference.

FIELD OF THE INVENTION

The invention relates generally to the field of imaging systems, andmore particularly to the imaging of objects. More specifically, theinvention relates to an improvement in an apparatus and method enablingdepth sectioned fluorescence imaging in a turbid medium, such as humanor animal tissue, in such a manner as to substantially minimize theexcitation radiation from reaching the detection beam path.

BACKGROUND OF THE INVENTION

It is well known in the art to use structured illumination to carry outfluorescence-based molecular imaging in turbid media. U.S. Publication2006/0184043 by Tromberg et al. (Tromberg) discloses a method forquantitative modulated imaging to perform depth sectioned reflectance ortransmission imaging in a turbid medium, such as human or animal tissue.The method is directed to steps of encoding periodic pattern ofillumination preferably with a fluorescent excitation wavelength whenexposing a turbid medium to the periodic pattern to providedepth-resolved discrimination of structures within the turbid medium;and reconstructing a non-contact three dimensional image of thestructure within a turbid medium. As a result, Tromberg states that widefield imaging, separation of the average background optical propertiesfrom the heterogeneity components from a single image, separation ofsuperficial features from deep features based on selection of spatialfrequency of illumination, or qualitative and quantitative structure,function and composition information may be extracted from spatiallyencoded data. However, Tromberg does not teach how to minimize theexcitation radiation from reaching the detection beam path.

U.S. Publication 2003/0010930 by Thorwirth discloses an arrangement forreading out the fluorescent radiation of specimen carriers with aplurality of individual specimens which for purposes of excitingfluorescent radiation in selected individual specimens comprises aswitchable electro-optical matrix for generating illumination which islimited in a spatially defined manner. An arrangement is disclosed forreading out the fluorescent radiation of selected individual specimensof multispecimen carriers having a switchable electro-optical matrix forgenerating illumination which is limited in a spatially defined manner,an optical system for imaging the electro-optical matrix on the specimencarrier, and a high-sensitivity photoreceiver for integral measurementof the fluorescent radiation of the excited individual specimens of thespecimen carrier. Thorwirth discloses a spatially differentiatedillumination of a specimen carrier with a plurality of specimens usingan electro-optical matrix which minimizes the proportion of excitationradiation contributing to the fluorescence signal in high-resolutionimaging. The electro-optical matrix and the specimen carrier areinclined relative to the optical axis of the optical system and aresubject to a Scheimpflug condition. The angles of inclination of theelectro-optical matrix and of the specimen carrier are selected suchthat the excitation radiation imaged by the light source unit on thespecimen carrier is reflected in such a way that essentially noexcitation radiation reaches the detection beam path. However, Thorwirthdoes not teach how to adapt the disclosed arrangement to enable depthsectioned fluorescence imaging in a turbid medium.

SUMMARY OF THE INVENTION

An object of the present invention is to provide an apparatus and methodfor enabling analytical imaging of an object.

Another object of the present invention is to provide an improvement insuch an apparatus and method for enabling depth sectioned fluorescenceimaging in a turbid medium, such as human or animal tissue, in such amanner as to substantially minimize the excitation radiation fromreaching the detection beam path. Minimizing the excitation radiationthat reaches the detection beam path both minimizes the potential forthat excitation radiation to cause background in the fluorescencesignal, and enables low cost emission filtration with high transmission.

These objects are given only by way of illustrative example, and suchobjects may be exemplary of one or more embodiments of the invention.Other desirable objectives and advantages inherently achieved by thedisclosed invention may occur or become apparent to those skilled in theart. The invention is defined by the claims.

According to one aspect of the present invention, there is provided animaging system for imaging an object. More specifically, there isprovided an improvement in an imaging system enabling depth sectionedfluorescence imaging in a turbid medium, such as human or animal tissue,in such a manner as to substantially minimize the excitation radiationfrom reaching the detection beam path. The imaging system includes anarrangement of the excitation radiation source such that the opticalaxis of the source is inclined relative to the optical axis of thecamera, the optical plane of the source and the optical plane of theobject are subject to a Scheimpflug condition provided by projectionoptics, and the angle of inclination of the source is selected such thatthe component of the excitation radiation incident upon the object thatis not absorbed by the object is scattered in such a way thatsubstantially minimizes excitation radiation from reaching the detectionbeam path.

One embodiment of the invention concerns an apparatus for quantitativemodulated fluorescence imaging to perform depth sectioned fluorescenceimaging of a turbid sample including a fluorescent turbid medium. Theapparatus includes projection optics, including a first optical axis, toexpose the turbid sample to a periodic pattern of excitation radiationto provide depth-resolved discrimination of fluorescent structureswithin the turbid medium; an image capture module, including a secondoptical axis and a detection beam path, to receive a data image from thesample; and a signal processor to transform the data image from thesample, spatially filter the transformed data image from the sample, andreconstruct the filtered, transformed data image from the sample. Theembodiment includes an arrangement whereby the first optical axis isinclined relative to the second optical axis; the projection opticsinclude an object plane and an image plane that are subject to aScheimpflug condition; and the projection optics has an angle ofinclination relative to an image plane of the apparatus, the angle ofinclination being selected such that the component of excitationradiation incident upon the sample that is not absorbed by the sample isscattered in such a way that substantially reduces excitation radiationfrom reaching the detection beam path. In another embodiment, theperiodic pattern of excitation radiation has periodicity in a directionperpendicular to a direction of a projection of the first optical axisonto the image plane, so that the phase of the periodic pattern ofexcitation radiation does not change with increasing depth into an imagespace.

One embodiment of a method according to the invention concernsquantitative modulated fluorescence imaging to perform depth sectionedfluorescence imaging of a turbid sample composed of a fluorescent turbidmedium. The method comprises using a computer to perform steps of:acquiring two or more fluorescence image sets from two or more sets ofprojection optics, whose optical axes have different angles ofinclination relative to an optical axis of an image capture module, toprovide coverage of regions shadowed, by sample topography, for any oneset of projection optics; and merging the two or more fluorescence imagesets.

Another embodiment of a method according to the invention also concernsa method for performing depth sectioned fluorescence imaging of a turbidsample including a fluorescent turbid medium. The method uses anapparatus for quantitative modulated fluorescence imaging, the apparatusincluding projection optics with a first optical axis, to expose theturbid sample to a periodic pattern of excitation radiation to providedepth-resolved discrimination of fluorescent structures within theturbid medium; an image capture module, including a second optical axisand a detection beam path, to receive a data image from the sample; anda signal processor to transform the data image from the sample,spatially filter the transformed data image from the sample, andreconstruct the filtered, transformed data image from the sample. Themethod includes steps of: inclining the first optical axis relative tothe second optical axis; providing in the projection optics an objectplane and an image plane that are subject to a Scheimpflug condition;and inclining the projection optics at an angle of inclination relativeto an image plane of the apparatus, the angle of inclination beingselected such that the component of excitation radiation incident uponthe sample that is not absorbed by the sample is scattered in such a waythat substantially reduces excitation radiation from reaching thedetection beam path. In another embodiment of the method, a further stepcomprises providing the periodic pattern of excitation radiation with aperiodicity in a direction perpendicular to a direction of theprojection of the first optical axis onto the image plane, so that thephase of the periodic pattern of excitation radiation does not changewith increasing depth into an image space.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features, and advantages of theinvention will be apparent from the following more particulardescription of the embodiments of the invention, as illustrated in theaccompanying drawings.

The elements of the drawings are not necessarily to scale relative toeach other.

FIG. 1 shows a partially cutaway perspective view of an exemplaryelectronic imaging system.

FIG. 2 shows a cutaway perspective view of components of the imagecapture module of the imaging system of FIG. 1 in accordance with afirst embodiment of the present invention wherein spatially modulatedexcitation radiation is delivered from direction “a” using anon-telecentric Scheimpflug lens system.

FIG. 3 shows a diagrammatic view of the spatial modulation grid used inthe image capture module of FIG. 2.

FIG. 4 shows a diagrammatic view of a plurality of spatial modulationgrids, each with a different spatial frequency, formed in series in aslider.

FIG. 5 shows a workflow diagram in accordance with a first method of thepresent invention.

FIG. 6 shows a workflow diagram of an exemplary method used in step 50 aof FIG. 5.

FIGS. 7A, 7B and 7C show cutaway diagrammatic views of the image capturemodule configured according to FIG. 2.

FIG. 8 shows diagrammatic views of the spatially modulated excitationradiation of FIGS. 7A, 7B and 7C.

FIGS. 9A, 9B, 9C, 9D, and 9E show optical simulation results generallyrepresenting the embodiment diagrammatically shown in FIG. 2.

FIG. 10 shows detail of the optical simulation results from FIGS. 9A and9E.

FIG. 11 shows a workflow diagram in accordance with a second method ofthe present invention.

FIG. 12 shows a cutaway perspective view of components of the imagecapture module of the imaging system of FIG. 1 in accordance with asecond embodiment of the present invention wherein spatially modulatedexcitation radiation is delivered from direction “a” using anon-telecentric Scheimpflug lens system.

FIGS. 13A, 13B and 13C show cutaway diagrammatic views of the imagecapture module con FIG. d according to FIG. 12.

FIG. 14 shows a cutaway perspective view of the image capture module ofFIG. 12 wherein spatially modulated excitation radiation is deliveredfrom direction “b” using a non-telecentric Scheimpflug lens system.

FIGS. 15A, 15B and 15C show cutaway diagrammatic views of the imagecapture module con FIG. d according to FIG. 14.

FIG. 16 shows a graphic representation of the workflow diagram of FIG.11.

FIG. 17 shows a cutaway perspective view of components of the imagecapture module of the imaging system of FIG. 1 in accordance with athird embodiment of the present invention wherein spatially modulatedexcitation radiation is delivered from direction “a” using a doublytelecentric Scheimpflug lens system.

FIGS. 18A, 18B, 18C, 18D, and 18E show optical simulation resultsgenerally representing the embodiment diagrammatically shown in FIG. 17.

FIG. 19 shows a summarized comparison of the depth-of-modulationresponse from the optical simulation results of FIGS. 9A, 9B, 9C, 9D,9E, 18A, 18B, 18C, 18D, and 18E.

FIG. 20 shows a summarized comparison of the DC-level response from theoptical simulation results of FIGS. 9A, 9B, 9C, 9D, 9E, 18A, 18B, 18C,18D, and 18E.

FIG. 21 shows a graphic representation of the workflow diagrams of FIGS.5 and 6.

FIG. 22 shows a cutaway perspective view of components of the imagecapture module of the imaging system of FIG. 1 in accordance with afourth embodiment of the present invention wherein spatially modulatedexcitation radiation is delivered from direction “a” using a doublytelecentric Scheimpflug zoom lens system con FIG. d for highmagnification.

FIGS. 23A, 23B and 23C show cutaway diagrammatic views of the imagecapture module con FIG. d according to FIG. 22.

FIG. 24 shows a cutaway perspective view of the image capture module ofFIG. 22 but instead con FIG. d for low magnification.

FIGS. 25A, 25B and 25C show cutaway diagrammatic views of the imagecapture module con FIG. d according to FIG. 24.

FIG. 26 shows a cutaway perspective view of components of anotherexemplary electronic imaging system in accordance with a fifthembodiment of the present invention wherein the optical plane of theexcitation source and the optical plane of the object are subject to aScheimpflug condition provided by projection optics, and the opticalplane of the object and the optical plane of the camera image are alsosubject to a Scheimpflug condition provided by imaging optics, wherebythe optical plane of the excitation source and the optical plane of thecamera image are orthogonal. and

FIG. 27 shows a cutaway perspective view of the electronic imagingsystem of FIG. 26.

DETAILED DESCRIPTION OF THE INVENTION

The invention has been described in detail with particular reference tocertain preferred embodiments thereof, but it will be understood thatvariations and modifications can be effected within the spirit and scopeof the invention. The following is a detailed description of thepreferred embodiments of the invention, reference being made to thedrawings in which the same reference numerals identify the same elementsof structure in each of the several figures.

FIG. 1 shows a partially cutaway perspective view of an exemplaryelectronic imaging system 1. The imaging system includes an excitationradiation source 10 for fluorescence excitation, an image capture module20 to receive the data image from a sample, a sample cabinet 30, and acommunication and computer control system 40. Source 10 includes a lampunit 12, for example a halogen or xenon lamp unit, and an excitationfilter wheel 14 containing a plurality of excitation filters 16 a, b,and c. Alternative excitation radiation sources known in the art includelamp sources employing an excitation filter slider, light emitting diodebased sources, and laser sources. Source 10 is optically coupled toimage capture module 20, for example by a randomized fiber optic bundle,not illustrated. Image capture module 20 delivers excitation radiation100 via projection optics to an optically transparent platen 120, whichis incorporated into a subject stage 121, and upon which is placed animmobilized subject, such as an anesthetized mouse 130. Aside from theexcitation radiation 100, image capture module 20 is substantiallyoptically sealed from ambient light. Sample cabinet 30 is alsosubstantially optically sealed from ambient light, and includes a door32. Communication and computer control system 40 communicates with imagecapture module 20 via a communication cable 41, and can include adisplay device 42, for example, a computer monitor.

FIG. 2 shows a cutaway perspective view of components of image capturemodule 20 in accordance with a first embodiment of the present inventionwherein spatially modulated excitation radiation is delivered fromdirection “a” using projection optics comprised of a non-telecentricScheimpflug lens system 115. The X-Y-Z coordinate system is defined 200.Excitation radiation 100 is transmitted through a one-dimensionalspatial modulation grid 105. The spatial modulation grid is coplanarwith the object plane of a non-telecentric Scheimpflug lens system 115.In the embodiment shown, the non-telecentric Scheimpflug lens systemincludes a single lens group as indicated; however, generally more thanone lens group may comprise a non-telecentric Scheimpflug lens system.The spatial modulation grid is configurable or movable to produce aplurality of phases that shift along the direction indicated by arrow165. Lens system 115 delivers the spatially modulated excitationradiation through a beam path 110 to the top surface of the platen 120,which surface is coplanar with the image plane of lens system 115, i.e.,the X-Y plane. By definition, a Scheimpflug lens system forms an imageof an object whereby the object and image planes are not parallel toeach other, but are instead inclined with respect to each other. Theexamples used throughout this specification show object and image planes(such as at grid 105 and top surface of platen 120, respectively) thatare perpendicular with respect to each other; but in general theinclination of the object and image planes can be any arbitrary angle,including obtuse angles if folding mirrors are used in the Scheimpfluglens system. Upon reaching the platen surface, the spatially modulatedexcitation radiation 125 a, b, and c, propagates further into the spacebeyond the platen, i.e., into the image space depth, which is thepositive Z direction. The characteristics of the spatial profile of theexcitation radiation, such as the depth of modulation and DC level atvarious planes parallel to the X-Y image plane through the image spacedepth, depend on both the image forming properties of the lens system,such as the depth of focus, as well as the optical properties, such asthe turbidity, of the medium in the image space. An immobilized subject,such as a fluorophore-labeled anesthetized mouse 130, situated at theplaten surface fills the image space with a turbid medium and provides aspatially distributed fluorescence signal with spatial modulation inproportion to the spatially modulated excitation radiation through theimage space. The image space of the excitation Scheimpflug lens system115 is the object space of the fluorescence detection lens system. Thefluorescence signal is imaged through a beam path 135 by a detectionlens system including a detection lens 155 and a detection lens diopter145, onto a sensor in a digital camera 160, such as a thermoelectricallycooled charge coupled device camera. A folding mirror 140 inserted inthe detection beam path enables a compact layout of the image capturemodule. A plurality of emission filters 150 a, b, c, and d in anemission filter wheel 151 provides spectral selection of thefluorescence signal using an actuator 152, as well as rejection ofexcitation radiation from the sensor. The component of the excitationradiation from beam path 110 that is not absorbed by the mouse 130 onplaten 120 is scattered predominantly along a direction indicated by thearrow 102; so that the excitation radiation is scattered away from thedetection beam path, thereby minimizing the potential for thatexcitation radiation to cause background in the fluorescence signal.Furthermore, because cost of the emission filters is increased andfluorescence transmission of the emission filters is decreased withincreased rejection of the excitation radiation, the reflection of theexcitation radiation away from the detection beam path enables low costemission filtration with high transmission.

FIG. 3 shows a diagrammatic view of spatial modulation grid 105 used inimage capture module 20. In the embodiments described throughout, thespatial modulation grid includes an alternating periodic pattern oftransparent and non-transparent stripes, 1050 and 1051, respectively.The spatial modulation grid is oriented so that the alternation of theperiodic pattern of transparent and non-transparent stripes is along thedirection shown by arrow 165 in FIGS. 2 and 3, i.e., parallel to theimage plane, in this example the platen surface. Furthermore, thespatial modulation grid can be incrementally shifted or translated alongthe direction indicated by the arrow 165, by fractions of the spatialmodulation period 1052. Such translation can be used to produce aplurality of phases of the spatial modulation corresponding to aplurality of fluorescence images comprising a fluorescence image set,whereby one phase of spatial modulation is selected for eachfluorescence image in the fluorescence image set so as to perform depthsectioning. The translation of the spatial modulation grid may beachieved by a piezo-electronically driven actuator, not illustrated,wherein amplified voltage is applied to a piezoelectric crystal tochange its length, thereby providing highly accurate repositioning ofthe grid pattern.

The spatial modulation grid 105 may be formed by selective removal ofmaterial from a solid sheet of material, and may be simply a single gridwith a given spatial modulation frequency. Alternatively, as shown inFIG. 4, grids may be formed in, and selected by means of, a slider 205including a plurality of spatial modulation grids, each with a differentspatial frequency, for example “high” 206, “medium” 207, and “low” 208,in series. The different spatial frequencies enable differentresolutions of depth sectioning. The series of spatial modulation gridsmay be distributed along the direction indicated by the arrow 165 to bein the same direction as the direction of spatial modulation phaseshift, thereby simplifying the actuation means necessary to achieve bothspatial modulation phase shift as well as grid selection. Alternatively,the series of spatial modulation grids may be distributed along adirection different from the direction of spatial modulation phaseshift. In alternative embodiments, the spatial modulation grid may be anelectronically programmable electro-optic matrix, for example a liquidcrystal matrix or a digital micromirror matrix.

FIG. 5 shows a workflow diagram in accordance with a first method of thepresent invention. First, a fluorescence image set is acquired using aplurality of phases of spatially modulated excitation radiationdelivered from direction “a”, step 50 a. Second, depth sectioning isperformed based on the depth of modulation in the fluorescence images,step 60 a. The depth sectioning is performed by communication andcomputer control system 40 which includes a signal processor to Fouriertransform the data image of the sample, spatially filter the transformeddata image of the sample, and reconstruct the filtered, transformed dataimage of the sample.

FIG. 6 shows a workflow diagram of an exemplary method used in step 50 aof FIG. 5 wherein the plurality of phases includes three relativephases, specifically 0 degrees step 51 a, 120 degrees step 51 b, and 240degrees step 51 c, i.e., one-third steps of the spatial modulationperiod length. FIGS. 7A, 7B and 7C show cutaway diagrammatic views ofimage capture module 20. The perspective of the view is from directlybelow platen 120. FIGS. 7A, 7B and 7C show the spatially modulatedexcitation radiation 125 a, b, and c, respectively, whereby the relativephase of the spatial modulation is 0 degrees, 120 degrees, and 240degrees, respectively. X-Y-Z coordinate system 200 is shown. FIG. 8shows diagrammatic views of the spatially modulated excitation radiationof FIGS. 7A, 7B and 7C in the X-Y plane. The relative phase is shiftedby 0 degrees, 120 degrees, and 240 degrees in 125 a, b, and c,respectively.

FIGS. 9A, 9B, 9C, 9D, and 9E show optical simulation results generallyrepresenting the embodiment diagrammatically shown in FIG. 2. Theoptical simulation was executed using the TracePro® optical modelingsoftware from Lambda Research Corporation (www.lambdares.com). A spatialmodulation grid pitch of 2 mm, a biconvex lens with focal length 103 mmas the non-telecentric Scheimpflug lens system, an object plane-to-lensdistance of 194 mm, and an image plane-to-lens distance of 194 mm, wereused in the simulation. The optical simulation results include thespatially modulated excitation radiation patterns present at a series ofplanes, parallel to the X-Y image plane, distributed through the imagespace depth beyond the platen, i.e., in the positive Z direction. Seethe first five plots at the bottom of FIGS. 9A, 9B, 9C, 9D, and 9E fordepths of 0, 4, 8, 12 and 16 mm, respectively. Three different spatialmodulation phases are shown for each depth. See the three rows of plotsfor modulation phases of 0, 120 and 240 degrees. Whereas the diagrams ofthe spatially modulated excitation radiation patterns 125 a, b, and c ofFIGS. 7A, 7B and 7C show an undersized round excitation radiation beamintersecting the spatial modulation grid, the optical simulations wereperformed using an excitation radiation beam that fills the spatialmodulation grid so as to more clearly illustrate the distortion inherentto a non-telecentric Scheimpflug lens system. Specifically, thetransverse magnification exhibits a gradient in the Y direction, i.e.,along the direction parallel to the direction of the projection of theexcitation radiation propagation vector onto the X-Y image plane. Thetransverse magnification in the Y direction increases with increasingdistance away from the object plane of the non-telecentric Scheimpfluglens system. Furthermore, the transverse magnification also exhibits agradient along the Z direction, normal to the image plane; thetransverse magnification increases with increasing distance away fromthe X-Y image plane of the non-telecentric Scheimpflug lens system. Inthese simulations, the best focus is at 0 mm, defined as the platensurface. The simulations show that the depth of modulation of theexcitation radiation pattern decreases with increasing depth into theimage space beyond the platen surface; i.e., the pattern is going out offocus. These simulations were performed for the case where the medium inthe image space beyond the platen is not turbid; if the medium wereinstead turbid, then the turbidity would accelerate the decrease of thedepth of modulation of the excitation radiation pattern with increasingdepth into the image space beyond the platen surface. The profiles takenalong the X direction, i.e., along the direction perpendicular to thedirection of the projection of the excitation radiation propagationvector onto the image plane, further illustrate the decrease of thedepth of modulation, defined as the ratio of the amplitude of thespatial oscillation, i.e., AC, to the average, i.e., DC, level, of theexcitation radiation pattern with increasing depth into the image spacebeyond the platen surface. The profiles also illustrate the rapiddecrease in the DC level of the excitation radiation pattern withincreasing depth into the image space beyond the platen surface due tothe increase of the transverse magnification with depth, i.e., theexcitation radiation spreads out over a larger area. The simulationsalso show that the spatially modulated excitation radiation patternshifts in the positive Y direction, away from the object plane of thenon-telecentric Scheimpflug lens system, with increasing depth into theimage space beyond the platen surface. This shifting behavior isinherent to any Scheimpflug lens system and is due to abnormality of thepropagation vector of the excitation radiation with respect to the imageplane. This shifting behavior restricts the orientation of the spatialmodulation to be aligned with the X direction so that the phase of thespatial modulation does not change with increasing depth into the imagespace beyond the platen surface. This shifting behavior also restrictsthe field of view of depth sectioning to that between the maximum Yextent of the X-Y image plane and the minimum Y extent of the deepestplane (shown by example as 16 mm deep).

FIG. 10 shows four plots of detail of the optical simulation resultsfrom FIGS. 9A and 9E. The profiles show the excitation radiation spatialmodulation, both at the image plane and at the plane 16 mm deep, in a 4mm range around the center of the pattern. The profiles show theexcitation radiation spatial modulation for the plurality of phases, byexample three phases. The vertical bars in the profiles representsampling points in the image plane and in the plane 16 mm deep. Theplots of excitation intensity vs. phase, which are the intersectionpoints of the vertical bars with the profiles corresponding to the threephases, show that at the sampling points, the depth of modulationdecreases with increasing depth into the image space beyond the platensurface. FIG. 10 illustrates use of an algorithm of a type familiar tothose skilled in the art to execute step 60 a, namely the point-by-pointanalysis of the depth of modulation to achieve point-by-point depthsectioning. For example, the algorithm disclosed by Tromberg wouldsuffice.

FIG. 11 shows a workflow diagram in accordance with a second method ofthe present invention, as practiced using the capture module 21 of FIG.12. First, a fluorescence image set is acquired using a plurality ofphases of spatially modulated excitation radiation delivered fromdirection “a”, step 50 a. Second, depth sectioning is performed based onthe depth of modulation in the fluorescence images, step 60 a. Third, afluorescence image set is acquired using a plurality of phases ofspatially modulated excitation radiation delivered from direction “b”,step 50 b. Fourth, depth sectioning is performed based on the depth ofmodulation in the fluorescence images, step 60 b. Last, the outputs ofsteps 60 a and 60 b are merged to fill in regions shadowed fromspatially modulated excitation light delivered from direction “a” byusing spatially modulated excitation light delivered from direction “b”.

FIG. 12 shows a cutaway perspective view of components of the imagecapture module 21 in accordance with a second embodiment of the presentinvention wherein spatially modulated excitation radiation is deliveredfrom direction “a” using projection optics including a non-telecentricScheimpflug lens system 115. This embodiment is similar to theembodiment shown in FIG. 2, however an additional spatial modulationgrid 106 and non-telecentric Scheimpflug lens system 116,minor-symmetric to spatial modulation grid 105 and non-telecentricScheimpflug lens system 115 across the X-Z plane bisecting the platen120, are included for delivery of excitation radiation from direction“b” in an additional step. FIGS. 13A, 13B and 13C show cutawaydiagrammatic views of the image capture module 21 configured accordingto FIG. 12. FIGS. 13A, 13B and 13C are similar to FIGS. 7A, 7B and 7C.

FIG. 14 shows a cutaway perspective view of the image capture module 21of FIG. 12 wherein spatially modulated excitation radiation is deliveredfrom direction “b” using projection optics including a non-telecentricScheimpflug lens system 116. Excitation radiation 101 is transmittedthrough a one-dimensional spatial modulation grid 106. The spatialmodulation grid is located at the object plane of a non-telecentricScheimpflug lens system 116. The spatial modulation grid is configurableor movable to produce a plurality of phases that shift along thedirection indicated by arrow 166. Lens system 116 delivers the spatiallymodulated excitation radiation through a beam path 111 to the surface ofthe platen 120 located at the image plane of the lens system, i.e., theX-Y plane. Upon reaching the platen surface, the spatially modulatedexcitation radiation 126 a, b, and c, propagates further into the spacebeyond the platen, i.e., into the image space depth, which is thepositive Z direction. The image space of excitation Scheimpflug lenssystem 116 is the object space of the fluorescence detection lenssystem. The fluorescence signal is imaged through a beam path 136 by thedetection lens system described previously. The excitation radiation isreflected along a direction indicated by the arrow 103; therefore, theexcitation radiation is reflected away from the detection beam path,thereby minimizing the potential for that excitation radiation to causebackground in the fluorescence signal. FIGS. 15A, 15B and 15C showcutaway diagrammatic views of the image capture module 21 configuredaccording to FIG. 14. FIGS. 15A, 15B and 15C are mirror-symmetric toFIGS. 13A, 13B and 13C across the X-Z plane bisecting the platen 120.

FIG. 16 shows a graphic representation of the workflow diagram of FIG.11. The section representing steps 50 a and 60 a shows spatiallymodulated excitation radiation pattern 125 a, b, and c delivered fromdirection “a” through beam path 110 to a hypothetical subject 131.Because the subject has topography, a shadow 132 a is cast, therebypreventing depth sectioning in the shadowed region. The sectionrepresenting steps 50 b and 60 b shows spatially modulated excitationradiation pattern 126 a, b, and c delivered from direction “b” throughbeam path 111 to the hypothetical subject 131. A different shadow 132 bis cast, but the region corresponding to shadow 132 a is herebyilluminated. The section representing step 70 shows the coverage ofbi-directional spatially modulated excitation radiation 127 a, b, and c.The merger of the outputs of steps 60 a and 60 b provides full coverageof the subject area, in effect eliminating shadows.

FIG. 17 shows a cutaway perspective view of components of the imagecapture module 22 in accordance with a third embodiment of the presentinvention wherein spatially modulated excitation radiation is deliveredfrom direction “a” using projection optics including a doublytelecentric Scheimpflug lens system 215. This embodiment is similar tothe embodiment shown in FIG. 12, except the non-telecentric Scheimpfluglens systems 115 and 116 have been replaced with doubly telecentricScheimpflug lens systems 215 and 216, respectively. In the embodimentshown, the doubly telecentric Scheimpflug lens systems each include twolens groups as indicated; however, generally more than two lens groupsmay comprise a doubly telecentric Scheimpflug lens system. By “doublytelecentric”, it is meant that the lens system provides both objectspace telecentricity and image space telecentricity. The lens systemdelivers the spatially modulated excitation radiation through a beampath 210 to the surface of the platen 120 located at the image plane ofthe lens system, i.e., the X-Y plane. Upon reaching the platen surface,the spatially modulated excitation radiation 225 a, b, and c, propagatesfurther into the space beyond the platen, i.e., into the image spacedepth, which is the positive Z direction. The image space of theexcitation Scheimpflug lens system is the object space of thefluorescence detection lens system, whereby the fluorescence signal isimaged through a beam path 235 by the detection lens system describedpreviously. The excitation radiation is reflected along a directionindicated by the arrow 102; therefore, the excitation radiation isreflected away from the detection beam path, thereby minimizing thepotential for that excitation radiation to cause background in thefluorescence signal.

FIGS. 18A, 18B, 18C, 18D, and 18E show optical simulation resultsgenerally representing the embodiment diagrammatically shown in FIG. 17,i.e., an embodiment based on a doubly telecentric Scheimpflug lenssystem. The optical simulation was executed using TracePro opticalmodeling software from Lambda Research Corporation. A spatial modulationgrid pitch of 2 mm, a matched pair of achromatic doublet lenses with 100mm focal length (Edmund Optics, Inc., stock number NT49-390) as thedoubly telecentric Scheimpflug lens system, an object plane-to-lensdistance of 100 mm, an image plane-to-lens distance of 100 mm, and alens-to-lens separation of 194 mm, were used in the simulation. Theoptical simulation results include the spatially modulated excitationradiation patterns present at a series of planes, parallel to the X-Yimage plane, distributed through the image space depth beyond theplaten, i.e., in the positive Z direction. Three different spatialmodulation phases are shown for each depth. Whereas the diagrams of thespatially modulated excitation radiation pattern 225 a, b, and c show anundersized round excitation radiation beam intersecting the spatialmodulation grid, the optical simulations were performed using anexcitation radiation beam that fills the spatial modulation grid so asto more clearly illustrate the absence of distortion inherent to adoubly telecentric Scheimpflug lens system and to compare with theoptical simulation results shown in FIGS. 9A, 9B, 9C, 9D, and 9E.Specifically, the transverse magnification provided by the doublytelecentric Scheimpflug lens system does not exhibit a gradient in the Ydirection, i.e., along the direction parallel to the direction of theprojection of the excitation radiation propagation vector onto the X-Yimage plane, unlike the non-telecentric Scheimpflug lens system.Furthermore, the transverse magnification provided by the doublytelecentric Scheimpflug lens system does not exhibit a gradient alongthe Z direction, normal to the image plane, unlike the non-telecentricScheimpflug lens system. As in the simulations for the non-telecentricScheimpflug lens system shown in FIGS. 9A, 9B, 9C, 9D, and 9E, in thesesimulations, the best focus is at 0 mm, defined as the platen surface.Similarly to the simulations for the non-telecentric Scheimpflug lenssystem shown in FIGS. 9A, 9B, 9C, 9D, and 9E, these simulations showthat the depth of modulation of the excitation radiation patterndecreases with increasing depth into the image space beyond the platensurface; i.e., the pattern is going out of focus. As in the simulationsfor the non-telecentric Scheimpflug lens system shown in FIGS. 9A, 9B,9C, 9D, and 9E, these simulations were performed for the case where themedium in the image space beyond the platen is not turbid; if the mediumwere instead turbid, then the turbidity would accelerate the decrease ofthe depth of modulation of the excitation radiation pattern withincreasing depth into the image space beyond the platen surface.Similarly to FIGS. 9A, 9B, 9C, 9D, and 9E, the profiles taken along theX direction, i.e., along the direction perpendicular to the direction ofthe projection of the excitation radiation propagation vector onto theimage plane, further illustrate the decrease of the depth of modulation,defined as the ratio of the amplitude of the spatial oscillation, i.e.,AC, to the average, i.e., DC, level, of the excitation radiation patternwith increasing depth into the image space beyond the platen surface.Unlike the simulations for the non-telecentric Scheimpflug lens systemshown in FIGS. 9A, 9B, 9C, 9D, and 9E, however, these profilesillustrate little decrease in the DC level of the excitation radiationpattern with increasing depth into the image space beyond the platensurface due to the constancy of the transverse magnification with depth,i.e., the excitation radiation does not spread out over a larger area.Similarly to the simulations for the non-telecentric Scheimpflug lenssystem shown in FIGS. 9A, 9B, 9C, 9D, and 9E, these simulations alsoshow that the spatially modulated excitation radiation pattern shifts inthe positive Y direction, away from the object plane of the doublytelecentric Scheimpflug lens system, with increasing depth into theimage space beyond the platen surface. As described previously, thisshifting behavior also restricts the field of view of depth sectioningto that between the maximum Y extent of the X-Y image plane and theminimum Y extent of the deepest plane (shown by example as 16 mm deep).

FIG. 19 shows a summarized comparison of the depth-of-modulationresponse from the optical simulation results of FIGS. 9A, 9B, 9C, 9D,9E, 18A, 18B, 18C, 18D, and 18E. The depth of modulation was calculatedfrom sinusoidal fits to profiles from the optical simulations describedpreviously. It is advantageous to have a steep decline in depth ofmodulation with increasing depth into the image space beyond the platensurface in order to achieve high depth-sectioning resolution. Thecomparison shows that the modeled doubly telecentric Scheimpflug lenssystem exhibits a steeper decline of depth of modulation with increasingdepth into the image space beyond the platen surface than does themodeled non-telecentric Scheimpflug lens system. This is in large partbecause the focal length of the image lens of the modeled doublytelecentric Scheimpflug lens system is shorter than the modelednon-telecentric Scheimpflug lens system, so that the depth of focus ofthe modeled doubly telecentric Scheimpflug lens system is less than themodeled non-telecentric Scheimpflug lens system. The difference in thedepth-of-modulation response shown here is not inherently characteristicof the non-telecentric and doubly telecentric Scheimpflug lens systems.

FIG. 20 shows a summarized comparison of the DC-level response from theoptical simulation results of FIGS. 9A, 9B, 9C, 9D, 9E, 18A, 18B, 18C,18D, and 18E. The DC level was calculated from sinusoidal fits toprofiles from the optical simulations described previously. It isadvantageous to have a high DC level with increasing depth into theimage space beyond the platen surface in order to achieve highdepth-sectioning signal. The comparison shows that the modeled doublytelecentric Scheimpflug lens system maintains a high DC level withincreasing depth into the image space beyond the platen surface thandoes the modeled non-telecentric Scheimpflug lens system. This is due tothe constancy of the transverse magnification with depth inherentlycharacteristic of the doubly telecentric Scheimpflug lens system asopposed to the increase of the transverse magnification with depth ofinherently characteristic of the non-telecentric Scheimpflug lenssystem.

FIG. 21 shows a graphic representation of the workflow diagrams of FIGS.5 and 6. A physical three-dimensional distribution of fluorophores isshown at left. The distribution includes two parallel discs: one at 0mm, i.e., at the platen surface; the other raised 16 mm above the platensurface; and non-overlapping when viewed from the normal direction (theraised disc is shown northwest of the 0 mm disc). The simulated imagesrepresent step 50 a wherein a fluorescence image set is acquired using aplurality of phases of spatially modulated excitation radiationdelivered from direction “a”, wherein the plurality of phases includesthree relative phases, specifically 0 degrees step 51 a, 120 degrees 51b, and 240 degrees 51 c, i.e., one-third steps of the spatial modulationperiod length. The simulated images show that the depth of modulation ofthe 0 mm disc fluorescence is greater than the depth of modulation ofthe 16 mm disc fluorescence. The depth of modulation graph of FIG. 19represents step 60 a wherein depth sectioning is performed based on thedepth of modulation in the fluorescence images. The highdepth-of-modulation content in the image set is mapped to 0 mm depthwhereas the low depth-of-modulation content in the image set is mappedto 16 mm The output of step 60 a is the fluorescence tomographicreconstruction of the physical three-dimensional distribution offluorophores.

FIG. 22 shows a cutaway perspective view of components of the imagecapture module 23 of the imaging system 1 in accordance with a fourthembodiment of the present invention wherein spatially modulatedexcitation radiation is delivered from direction “a” using projectionoptics including a doubly telecentric Scheimpflug zoom lens system 315configured for high magnification. This embodiment is similar to theembodiment shown in FIG. 17, except the doubly telecentric Scheimpfluglens systems 215 and 216 have been replaced with doubly telecentricScheimpflug zoom lens systems 315 and 316, respectively. In theembodiment shown, the doubly telecentric Scheimpflug zoom lens systemseach include two lens groups as indicated; however, generally more thantwo lens groups may comprise a doubly telecentric Scheimpflug zoom lenssystem. One of ordinary skill in the art will understand that aplurality of doubly-telecentric fixed-focal lens systems providingdifferent magnifications would provide equivalent benefits as a doublytelecentric zoom lens system. The lens system delivers the spatiallymodulated excitation radiation through a beam path 310 to the surface ofthe platen 120 located at the image plane of the lens system, i.e., theX-Y plane. Upon reaching the platen surface, the spatially modulatedexcitation radiation 325 a, b, and c, propagates further into the spacebeyond the platen, i.e., into the image space depth, which is thepositive Z direction. The image space of the excitation Scheimpflug lenssystem is the object space of the fluorescence detection lens system,whereby the fluorescence signal is imaged through a beam path 335 by thedetection lens system described previously. The excitation radiation isreflected along a direction indicated by the arrow 102; therefore, theexcitation radiation is reflected away from the detection beam path,thereby minimizing the potential for that excitation radiation to causebackground in the fluorescence signal. FIGS. 23A, 23B and 23C showcutaway diagrammatic views of the image capture module 23 configuredaccording to FIG. 22. FIGS. 23A, 23B and 23C are similar to FIGS. 7A, 7Band 7C, except the spatial frequency of the excitation radiationmodulation has decreased due to the high magnification configuration ofthe zoom lens system. The zoom lens system provides an alternative meansfor adjusting the spatial frequency of the excitation radiationmodulation compared to that shown in FIG. 4.

FIG. 24 shows a cutaway perspective view of the image capture module 23of FIG. 22 but instead configured for low magnification. The lens systemdelivers the spatially modulated excitation radiation through a beampath 312 to the surface of the platen 120 located at the image plane ofthe lens system, i.e., the X-Y plane. Upon reaching the platen surface,the spatially modulated excitation radiation 327 a, b, and c, propagatesfurther into the space beyond the platen, i.e., into the image spacedepth, which is the positive Z direction. The image space of theexcitation Scheimpflug lens system is the object space of thefluorescence detection lens system, whereby the fluorescence signal isimaged through a beam path 337 by the detection lens system describedpreviously. The excitation radiation is reflected along a directionindicated by the arrow 102; therefore, the excitation radiation isreflected away from the detection beam path, thereby minimizing thepotential for that excitation radiation to cause background in thefluorescence signal. FIGS. 25A, 25B and 25C show cutaway diagrammaticviews of the image capture module 23 configured according to FIG. 24.FIGS. 25A, 25B and 25C are similar to FIGS. 23A. 23B and 23C, except thespatial frequency of the excitation radiation modulation has increaseddue to the low magnification configuration of the zoom lens system.

FIGS. 26 and 27 show cutaway perspective views of components of anotherexemplary electronic imaging system in accordance with a fifthembodiment of the present invention. The optical plane of the excitationsource and the optical plane of the object are subject to a Scheimpflugcondition provided by projection optics. The optical plane of the objectand the optical plane of the camera image are also subject to aScheimpflug condition provided by imaging optics. As a result, theoptical plane of the excitation source and the optical plane of thecamera image are orthogonal. Excitation radiation 2100 is transmittedthrough a one-dimensional spatial modulation grid 2105. The spatialmodulation grid is located at the object plane of a Scheimpflug lenssystem 2115. In the embodiment shown, the Scheimpflug lens systemincludes a single lens group as indicated; however, generally more thanone lens group may comprise a Scheimpflug lens system. The Scheimpfluglens system may be either non-telecentric or telecentric as describedpreviously. The spatial modulation grid is configurable to produce aplurality of phases that shift along the direction indicated by arrow2165. The lens system delivers the spatially modulated excitationradiation through a beam path 2110 to the surface of an immobilizedsubject, such as an anesthetized human patient 2130 undergoing surgerywho has been administered a fluorescent probe, located at the imageplane of the lens system, i.e., the X-Y plane. By definition, aScheimpflug lens system forms an image of an object whereby the objectand image planes are not parallel to each other, but are insteadinclined with respect to each other. Upon reaching the subject surface,the spatially modulated excitation radiation 2125 a, b, and c,propagates further into the space beyond the object surface, i.e., intothe image space depth, which is the positive Z direction. Thecharacteristics of the spatial profile of the excitation radiation, suchas the depth of modulation and DC level, at the various planes, parallelto the X-Y image plane, through the image space depth depend on both theimage forming properties of the lens system, such as the depth of focus,as well as the optical properties, such as the turbidity, of the mediumin the image space. The immobilized subject 2130 fills the image spacewith a turbid medium and provides a spatially distributed fluorescencesignal with spatial modulation in proportion to the spatially modulatedexcitation radiation through the image space. The image space of theexcitation Scheimpflug lens system is the object space of thefluorescence detection Scheimpflug lens system. The fluorescence signalis imaged through a beam path 2135 by a detection Scheimpflug lenssystem including a detection lens 2155, onto a sensor in a digitalcamera 2160, such as a thermoelectrically cooled charge coupled devicecamera, and an emission filter wheel (not shown), containing a pluralityof emission filters provides spectral selection of the fluorescencesignal as well as rejection of excitation radiation from the sensor. Theexcitation radiation is reflected along a direction indicated by thearrow 2102; therefore, the excitation radiation is reflected away fromthe detection beam path, thereby minimizing the potential for thatexcitation radiation to cause background in the fluorescence signal. Theorthogonality of the optical plane of the excitation source and theoptical plane of the camera image substantially minimizes excitationradiation from reaching the detection beam path. Furthermore, becausecost of the emission filters is increased and fluorescence transmissionof the emission filters is decreased with increased rejection of theexcitation radiation, the reflection of the excitation radiation awayfrom the detection beam path enables low cost emission filtration withhigh transmission. The detected image of the fluorescent signal isdisplayed on display device 2042, as useful for fluorescent image guidedsurgery. The Scheimpflug arrangement of both the fluorescence excitationoptics and the fluorescence imaging optics enables an accessible objectspace, such as would be desirable for fluorescence image guided surgeryas shown by surgeon's hands 2044.

PARTS LIST

-   1 exemplary electronic imaging system-   10 excitation radiation source-   12 lamp unit-   14 excitation filter wheel-   16 a, b, c excitation filters-   20 image capture module-   21 image capture module-   22 image capture module-   23 image capture module-   30 sample cabinet-   32 door-   40 communications and computer control system-   41 communication cable-   42 display device or monitor-   50 a, b step-   51 a, b, c step-   60 a, b step-   70 step-   100 excitation radiation from direction “a”-   101 excitation radiation from direction “b”-   102 direction of reflection of excitation radiation from direction    “a”-   103 direction of reflection of excitation radiation from direction    “b”-   105 spatial modulation grid-   106 spatial modulation grid-   110 beam path of spatially modulated excitation radiation delivered    from direction “a”-   111 beam path of spatially modulated excitation radiation delivered    from direction “b”-   115 non-telecentric Scheimpflug lens system-   116 non-telecentric Scheimpflug lens system-   120 optically transparent platen-   121 subject stage-   125 a, b, c spatially modulated excitation radiation pattern    delivered from direction “a”-   126 a, b, c spatially modulated excitation radiation pattern    delivered from direction “b”-   127 a, b, c coverage of bi-directional spatially modulated    excitation radiation-   130 anesthetized mouse-   131 hypothetical subject-   132 a, b shadow-   135 beam path of fluorescence detection-   136 beam path of fluorescence detection-   140 folding mirror-   145 detection lens diopter-   150 a, b, c, d emission filters-   151 emission filter wheel-   152 emission filter wheel actuator    -   155 detection lens    -   160 digital camera    -   165 direction to produce spatial phase shift-   166 direction to produce spatial phase shift-   200 X-Y-Z coordinate system-   205 slider with plurality of spatial modulation grids-   206 spatial modulation grid with “high” spatial modulation frequency-   207 spatial modulation grid with “medium” spatial modulation    frequency-   208 spatial modulation grid with “low” spatial modulation frequency-   210 beam path of spatially modulated excitation radiation delivered    from direction “a”-   215 doubly telecentric Scheimpflug lens system-   216 doubly telecentric Scheimpflug lens system-   225 a, b, c spatially modulated excitation radiation pattern    delivered from direction “a”-   235 beam path of fluorescence detection-   310 beam path of spatially modulated excitation radiation delivered    from direction “a”-   312 beam path of spatially modulated excitation radiation delivered    from direction “a”-   315 doubly telecentric Scheimpflug zooming lens system-   316 doubly telecentric Scheimpflug zooming lens system-   325 a, b, c spatially modulated excitation radiation pattern    delivered from direction “a”-   327 a, b, c spatially modulated excitation radiation pattern    delivered from direction “a”-   335 beam path of fluorescence detection-   337 beam path of fluorescence detection-   1050 transparent stripes-   1051 non-transparent stripes-   1052 spatial modulation period-   2042 display device or monitor-   2044 surgeon's hands-   2100 excitation radiation-   2102 direction of reflection of excitation radiation-   2105 spatial modulation grid-   2110 beam path of spatially modulated excitation radiation-   2115 Scheimpflug excitation lens system-   2125 a, b, c spatially modulated excitation radiation pattern-   2130 anesthetized human patient-   2165 direction to produce spatial phase shift-   2135 beam path of fluorescence detection-   2155 Scheimpflug detection lens system-   2160 digital camera

What is claimed is:
 1. An apparatus for quantitative modulatedfluorescence imaging to perform depth sectioned fluorescence imaging ofa turbid sample including a fluorescent turbid medium, the apparatusincluding projection optics, including a first optical axis, to exposethe turbid sample to a periodic pattern of excitation radiation toprovide depth-resolved discrimination of fluorescent structures withinthe turbid medium; an image capture module, including a second opticalaxis and a detection beam path, to receive a data image from the sample;and a signal processor to transform the data image from the sample,spatially filter the transformed data image from the sample, andreconstruct the filtered, transformed data image from the sample; theimprovement comprising: an arrangement whereby the first optical axis isinclined relative to the second optical axis; the projection opticsincluding an object plane and an image plane that are subject to aScheimpflug condition; the periodic pattern of excitation radiationhaving periodicity in a direction perpendicular to a direction of aprojection of the first optical axis onto the image plane, so that thephase of the periodic pattern of excitation radiation does not changewith increasing depth into an image space; and the projection opticshaving an angle of inclination relative to an image plane of theapparatus, the angle of inclination being selected such that thecomponent of excitation radiation incident upon the sample that is notabsorbed by the sample is scattered in such a way that substantiallyreduces excitation radiation from reaching the detection beam path. 2.The apparatus of claim 1, wherein the projection optics comprise one ormore non-telecentric Scheimpflug lens systems.
 3. The apparatus of claim2, wherein the projection optics comprise two non-telecentricScheimpflug lens systems that are mirror-symmetric.
 4. The apparatus ofclaim 2, wherein the one or more non-telecentric Scheimpflug lenssystems are zoomable.
 5. The apparatus of claim 2, wherein the one ormore non-telecentric Scheimpflug lens systems include a plurality offixed-focal lens systems, each providing a different magnification. 6.The apparatus of claim 1, wherein the projection optics comprise one ormore Scheimpflug lens systems providing object space telecentricity. 7.The apparatus of claim 6, wherein the projection optics comprise twoScheimpflug lens systems providing object space telecentricity that aremirror-symmetric.
 8. The apparatus of claim 6, wherein the one or moreScheimpflug lens systems providing object space telecentricity arezoomable.
 9. The apparatus of claim 6, wherein the one or moreScheimpflug lens systems providing object space telecentricity include aplurality of fixed-focal lens systems each providing a differentmagnification.
 10. The apparatus of claim 1, wherein the projectionoptics comprise one or more Scheimpflug lens systems providing imagespace telecentricity.
 11. The apparatus of claim 10, wherein theprojection optics comprise two Scheimpflug lens systems providing imagespace telecentricity that are mirror-symmetric.
 12. The apparatus ofclaim 10, wherein the Scheimpflug lens systems providing image spacetelecentricity are zoomable.
 13. The apparatus of claim 10, wherein theone or more Scheimpflug lens systems providing image spacetelecentricity include a plurality of fixed-focal lens systems eachproviding a different magnification.
 14. The apparatus of claim 1,wherein the projection optics comprise one or more doubly telecentricScheimpflug lens systems.
 15. The apparatus of claim 14, wherein theprojection optics comprise two doubly telecentric Scheimpflug lenssystems that are mirror-symmetric.
 16. The apparatus of claim 14,wherein the doubly telecentric Scheimpflug lens system(s) is (are)zoomable.
 17. The apparatus of claim 1, further comprising detectionoptics including an object plane and an optical plane of a camera imagethat are subject to a Scheimpflug condition.
 18. The apparatus of claim1, further comprising image detection optics including a first opticalplane of the sample and a second optical plane of images captured by theimage capture module, the first and second optical planes being subjectto a Scheimpflug condition.
 19. An apparatus for quantitative modulatedfluorescence imaging to perform depth sectioned fluorescence imaging ofa turbid sample including a fluorescent turbid medium, the apparatusincluding projection optics, including a first optical axis, to exposethe turbid sample to a periodic pattern of excitation radiation toprovide depth-resolved discrimination of fluorescent structures withinthe turbid medium; an image capture module, including a second opticalaxis and a detection beam path, to receive a data image from the sample;and a signal processor to transform the data image from the sample,spatially filter the transformed data image from the sample, andreconstruct the filtered, transformed data image from the sample; theimprovement comprising: an arrangement whereby the first optical axis isinclined relative to the second optical axis; the projection opticsincluding an object plane and an image plane that are subject to aScheimpflug condition; and the projection optics having an angle ofinclination relative to an image plane of the apparatus, the angle ofinclination being selected such that the component of excitationradiation incident upon the sample that is not absorbed by the sample isscattered in such a way that substantially reduces excitation radiationfrom reaching the detection beam path.
 20. A method for quantitativemodulated fluorescence imaging to perform depth sectioned fluorescenceimaging of a turbid sample composed of a fluorescent turbid medium,comprising using a computer to perform steps of: acquiring two or morefluorescence image sets from two or more sets of projection optics,whose optical axes have different angles of inclination relative to anoptical axis of an image capture module, to provide coverage of regionsshadowed, by sample topography, for any one set of projection optics;and merging the two or more fluorescence image sets.
 21. A method forperforming depth sectioned fluorescence imaging of a turbid sampleincluding a fluorescent turbid medium, using an apparatus forquantitative modulated fluorescence imaging, the apparatus includingprojection optics with a first optical axis, to expose the turbid sampleto a periodic pattern of excitation radiation to provide depth-resolveddiscrimination of fluorescent structures within the turbid medium; animage capture module, including a second optical axis and a detectionbeam path, to receive a data image from the sample; and a signalprocessor to transform the data image from the sample, spatially filterthe transformed data image from the sample, and reconstruct thefiltered, transformed data image from the sample, comprising: incliningthe first optical axis relative to the second optical axis; providing inthe projection optics an object plane and an image plane that aresubject to a Scheimpflug condition; and inclining the projection opticsat an angle of inclination relative to an image plane of the apparatus,the angle of inclination being selected such that the component ofexcitation radiation incident upon the sample that is not absorbed bythe sample is scattered in such a way that substantially reducesexcitation radiation from reaching the detection beam path.
 22. Themethod of claim 21, further comprising a step of providing the periodicpattern of excitation radiation with a periodicity in a directionperpendicular to a direction of the projection of the first optical axisonto the image plane, so that the phase of the periodic pattern ofexcitation radiation does not change with increasing depth into an imagespace.